Summary Dengue virus (DENV) infection causes major health problems with increasing incidence in endemic regions. Dengue fever, caused by any of the four DENV serotypes (DENV1-4), initially presents with non-specific symptoms of febrile illnesses, thus clinical diagnosis is often inaccurate. Early accurate diagnosis is important to monitor patients for severe complications due to internal hemorrhage and shock, which can be fatal without appropriate clinical management. Current DENV diagnostic methods either require specialized laboratory facilities, or have poor sensitivity in the acute phase. Antibody detection in the convalescent phase has poor specificity due to cross-reactivity with other flaviviruses. Nucleic acid amplification testing (NAAT) provides high sensitivity and specificity, but current technologies are incompatible with peripheral low resource healthcare facilities that serve the majority of affected patients. The goal of this R21 project is to develop an initial prototype for a portable, inexpensive, robust, easy to use, integrated nucleic acid testing device consisting of a disposable cartridge and an inexpensive, compact, battery operated instrument that enables early, rapid, sensitive, and specific diagnosis of acute DENV infection. We have already developed such an integrated prototype device for diagnosis of pulmonary tuberculosis from sputum. For diagnosis of DENV infection from whole blood, we have established proof of principle for processes required during sample preparation, amplification, and detection. Through the proposed project, we will further develop and optimize these processes, and then incorporate them into a cartridge and instrument prototype that leverages components of our existing NAAT device, but in a simpler format. Aim 1 entails optimization of processes for plasma separation, sample preparation, reverse transcription coupled to isothermal loop-mediated amplification (RT-LAMP), and visual lateral flow-based detection. In Aim 2 we will develop a prototype cartridge and instrument that integrates and automates these processes. Using the prototype device, we will demonstrate detection of all four DENV serotypes spiked into whole blood. Successful completion of this project entails demonstrating that the device can detect DENV1-4 in blood with limit of detection comparable to the FDA-cleared DENV1-4 real time RT-PCR assay developed by the Centers for Disease Control and prevention (CDC). In future efforts, once feasibility has been established, we will advance the initial prototype to a final version of the device, conduct full analytical validation and a field evaluation study, with the goal to obtain FDA clearance. Our long term goal is also to expand the multiplexing capabilities to enable differential diagnosis of Zika, dengue, and chikungunya viruses.